Fig. 3. Zebrafish models of gdf11 loss of function exhibit craniofacial and body axis patterning defects. (a) Overview of the gdf11 mutants generated via CRISPR/Cas9 gene editing (b–d) Alcian and Alizarin staining of the 7-dpf larval head skeleton labels cartilage (blue) and bone (red) elements. From the ventral aspect, Meckel’s cartilage (m) in the wild-type larval fish (b) extends rostrally beyond the ethmoid plate of the upper jaw (e, red dotted line delineates the rostral-most edge), the bilateral ceratohyal elements (ch) meet at the midline in a constrained angle of articulation (yellow dotted lines), and the opercular bone (op, red dotted circle) is ossified in with a broadening flare at its distal end. gdf11 mutants (c, d) exhibit defects in the alignment of upper and jaw elements, in the angle of ch articulation, and the morphology of the op with a more severe phenotype observed in the late truncating allele (d). (e, f) Upper and lower jaw element alignment are visualized again in sagittal sections of hematoxylin and eosin (H & E) stained 7-dpf wild-type (e) and gdf11 mutant (f) larvae, in which the ethmoid plate protrudes beyond the rostral limit of Meckel’s cartilage. (g, h) Six-month gdf11 mutant (h) rostral length measured from the anterior edge of the eye to the tip of the nose (white arrow) is 15% longer than in stage-matched wild-type (g; p = 0.0007) while the dorsoventral thickness of the head posterior to the eye (white double arrowhead, also marked in i,j) is an average of 15% less (p = 0.001) than in wild-type. (i, j) Regular anterior–posterior arrangements of body segments are visible on the lateral exterior or the juvenile fish (shown at 2 months in i and j), with eight such segments (white dotted lines) falling between the pectoral and pelvic (p) fins. One additional segment is noted in gdf11 mutants (j, white, and red dotted lines). N ≥ 8 for each group; scale bars: (b–f) 250 µm; (g–j) 1 mm.
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